Composition for treatment of tuberculosis

ABSTRACT

The invention relates to a pharmaceutical composition comprising a compound preventing EthR from binding to the ethA promoter, for example a compound of formula 1 wherein R 1  is optionally substituted phenyl or optionally substituted pyridyl; R 2  (CH 2 ) n  wherein n is 1, 2, 3 or 4; R 3  is CH 3 (CH 2 ) m  wherein m is 0, 1, 2 or 3; X 1  is O, S, NH, N(CH 3 ) or CH 2 ; and X 2  is O, S or NH; in particular 2-phenylethyl butyrate, and a thioamide or thiourea of formula 2 wherein R 4  is optionally substituted phenyl, optionally substituted pyridyl, optionally substituted indolyl, —NR 7 R 8 ; or —NH—N═CH—R 9 ; and substituents R 5  to R 9  have the meanings indicated in the description, in particular ethionamide. The pharmaceutical composition is useful, e.g., in the treatment of multidrug-resistant tuberculosis.

FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions useful in thetreatment of tuberculosis and related diseases.

BACKGROUND OF THE INVENTION

Up to 9 million people contract tuberculosis every year and 50 millionpeople are presently infected with Mycobacterium tuberculosis resistantto both first-line drugs isoniazid and rifampicin (WHO, Fact sheet No.104, March 2007). Ethionamide (2-ethylthioiso-nicotinamide,2-ethylpyrimidine-4-carbothioamide), a structural analogue of isoniazid,is currently the last line of defence in the treatment ofmulti-drug-resistant tuberculosis (MDR-TB). During 35 years of itsclinical use, ethionamide has fortunately elicited littlecross-resistance with isoniazid as both prodrugs have to be activated bydifferent mycobacterial enzymes to develop their antimicrobial activity.Yet, ethionamide continues to be prescribed at hepatotoxic doses as aconsequence of EthR repressing ethA, the monooxigenase that catalysesactivation of the prodrug ethionamide into an anti-mycobacterialnicotinamide adenine dinucleotide derivative. Up to a 1 g/day arerequired for an acceptable concentration in blood (Holdiness, M. R.,Clin Pharmacokinet 9, 511-44 (1984)), which is associated with severeside-effects including neurotoxicity and fatal hepatotoxicity.

SUMMARY OF THE INVENTION

The invention relates to a pharmaceutical composition comprising acompound preventing EthR from binding to the ethA promoter and athioamide or a thiourea. In particular the invention relates to such acomposition comprising a compound of formula 1

wherein R¹ is optionally substituted phenyl or optionally substitutedpyridyl;

R² is (CH₂), wherein n is 1, 2, 3 or 4;

R³ is CH₃(CH₂)_(m) wherein m is 0, 1, 2 or 3;

X¹ is O, S, NH, N(CH₃) or CH₂; and

X² is O, S or NH;

and a compound of formula 2

wherein R⁴ is optionally substituted phenyl, optionally substitutedpyridyl, optionally substituted indolyl, —NR⁷R⁸, or —NH—N═CH—R⁹;

R⁵ is hydrogen, C₁-C₆-alkyl, optionally substituted phenyl, optionallysubstituted pyridyl, or a sugar residue;

R⁶ is hydrogen or C₁-C₆-alkyl, or R⁵ and R⁶ together with the N-atom towhich they are bound are pyrrolidine, piperidine or morpholine;

R⁷ is hydrogen, C₁-C₆-alkyl, optionally substituted phenyl, optionallysubstituted pyridyl, or a sugar residue;

R⁸ is hydrogen or C₁-C₆-alkyl, or R⁷ and R⁸ together with the N-atom towhich they are bound are pyrrolidine, piperidine or morpholine; and

R⁹ is optionally substituted phenyl.

Most preferred is a composition comprising a compound of formula 1selected from 4-phenyl-2-butanone, benzyl acetate, 3-phenylpropylpropionate and 2-phenylethyl butyrate, in particular 2-phenylethylbutyrate, and a compound of formula 2 selected from ethionamide, isoxyl,N-arabinofuranosyl-N′-[ρ-(isoamyloxy)phenyl]-thiourea or thiacetazone,in particular ethionamide.

The invention likewise relates to the use of a composition comprising acompound preventing EthR from binding to the ethA promoter, e.g.2-phenylethyl butyrate, and a thiomide or a thiourea, e.g. ethionamide,in the treatment of tuberculosis and related diseases, and to a methodof treatment of tuberculosis and related diseases wherein a compositioncomprising a compound preventing EthR from binding to the ethA promoterand a thioamide or thiourea, e.g. ethionamide, is applied.

Furthermore the invention relates to a method of screening for compoundspreventing EthR from binding to the ethA promoter useful to increase thesensitivity of multidrug-resistant M. tuberculosis to a thioamide orthiourea, e.g. to ethionamide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. EthR-based synthetic gene network in mammalian cells.

(A) A gene fusion of ethR with the Herpes simplex-derived vp16transactivation domain is expressed under the control of the simianvirus 40 promoter (P_(SV40), plasmid pWW489) in HEK-293. The chimerictransactivator EthR-VP16 binds to its operator O_(ethR) therebyactivating transcription from the minimal Drosophila heat shock 70promoter (P_(hsp70min)), driving expression of human placental secretedalkaline phosphatase (seap, plasmid pWW491). In the presence of acell-permeable, non-cytotoxic inducer (IND), binding of EthR-VP16 to thepromoter is inhibited, thereby resulting in transcriptional silence(broken lines). Non cell-permeable or cytotoxic compounds areautomatically excluded from the hit list.

(B) Screening of a rationally designed compound library using theEthR-based gene network. 30,000 HEK-293 cells containing either theEthR-based gene network (pWW489 and pWW491, results shown with solidline) or an isogenic constitutive SEAP expression network (pWW35 andpWW37, results shown with dotted line) are cultivated for 48 h in thepresence of potential inducers (IND) prior to SEAP profiling. SEAPexpression was normalized to 100%. x-axis: concentration of testsubstance (mM); y-axis: SEAP production (%); PPP, 3-phenylpropylpropionate; PB, 4-phenyl-2-butanone; BA, benzyl acetate; PEB,2-phenylethyl butyrate.

(C) Clonal variability of HEK cells stably transduced with a retroviralvector encoding ethR-vp16 (pWW871, _(EthR)HEK). Individual clones(40,000 cells) are further transfected with plasmid pWW491 (see FIG. 1A)and cultivated at the indicated 2-phenlyethyl butyrate (PEB)concentrations for 48 h prior to quantifying SEAP production. x-axis:clone identification number.

(D) Clonal variability of HEK cells double transgenic for pWW871 andpWW491 (_(EthR)HEK-SEAP). 40,000 _(EthR)HEK-SEAP cells are cultivated atthe indicated 2-phenlyethyl butyrate (PEB) concentrations for 48 h priorto quantifying SEAP production. x-axis: clone identification number.

(E) Dose-response characteristics of the synthetic gene switch. 40,000double transgenic _(EthR)HEK-SEAP cells are cultivated at increasing2-phenylethyl butyrate (PEB) concentrations for 48 h prior toquantifying SEAP production. The different bars for each concentrationrepresent the different clones tested (clone identification numbers 14,23, 20).

(F) Reversibility of the synthetic gene circuit. 200,000 _(EthR)HEK-SEAPcells are alternately cultivated in the absence (−) or presence (+) of3.2 mM 2-phenlyethyl butyrate and SEAP production is profiled atindicated time points. At 72 h, the inducer status is reversed (arrow).x-axis: time (t)

FIG. 2. Validation of the inducer in bacteria and in a cell-free system

(A) Effect of 2-phenylethyl butyrate in E. coli. Upper panel: E.coliBL21(DE3), transformed with pWW488 and pWW856 (see FIG. 2C), is grown inthe presence of IPTG at the indicated 2-phenylethyl butyrate (PEB)concentrations for 5.5 h prior to analyzing the cells by FACS. Theoptical density at 600 nm (OD₆₀₀) after the growth period is indicatedas well. Lower panel: As a control, E.coli BL21(DE3) transformed withpWW856 alone are used in parallel. y-axis: percentage of gates cells.Gates: D, dead cells; E-D, cells with EthR dissociated from operator,EthR not present; E-B, cells with EthR bound to operator.

(B) Impact of 2-phenylethyl butyrate on the interaction between EthR andO_(ethR) in vitro. Biotinylated operator O_(ethR) immobilized onstreptavidin-agarose beads is incubated in the presence or absence of2-phenylethyl butyrate (PEB) at the indicated concentrations in a celllysate of E.coli BL21(DE3) transformed with pWW862(P_(T7)ethR-his₆-term) for production of hexahistidine(his₆)-taggedEthR. Following washing, his₆-tagged EthR is detected by a monoclonalanti-his₆ antibody coupled to horseradish peroxidase, resulting in theconversion of 3,3′,5,5′-tetramethylbenzidine to a colored formazanewhich is subsequently quantified via its absorption at 450 nm(OD₄₅₀)_(.) As negative controls, non-recombinant cell lysate is used(neg).

(C) Genetic setup of the screening system in bacteria. ethR-vp16 isexpressed under the control of the phage T7 promoter (P_(T7), plasmidpWW488). In the absence of the inducer, EthR-VP16 activates the chimericpromoter P_(ethR) and induces transcription of gfp (plasmid pWW856). Inthe presence of the inducer (IND), EthR-VP16 binding is inhibited,resulting in transcriptional silence.

(D) Characterization of the screening system. E.coli BL21(DE3),transformed with pWW488 and pWW856, are grown in the presence or absenceof IPTG for 5.5 h prior to FACS analysis. As controls, wild-type (wt)bacteria or bacteria transformed with pWW856 alone are used. The cellpopulations are grouped into three categories according to theirfluorescence levels (gates a, b, c).

FIG. 3. Specificity of the 2-phenylethyl butyrate activity.

To exclude that 2-phenylethyl butyrate indiscriminatingly modulatesprotein-DNA interactions the macrolide-dependent repressor-operatorconfiguration is challenged with this compound in an identical ELISAset-up as shown in FIG. 2B. The E-ETR interaction is insensitive to2-phenylethyl butyrate (PEB) and can exclusively be abolished in thepresence of erythromycin (EM, 5 μg/ml).

FIG. 4. Effect of 2-phenylethyl butyrate and ethionamide on M. bovis BCGand M. tuberculosis.

(A) Synergistic effect of 2-phenylethyl butyrate (PEB) and ethionamide(EA) on the growth inhibition of M. bovis BCG. A, B, C and D correspondto serial dilutions (10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵) of an M. bovis BCGsettling culture (OD₆₀₀: 0.6).

(B) Synergistic effect of 2-phenylethyl butyrate (PEB) and ethionamide(EA) on growth inhibition of M. tuberculosis H37Rv. A, B, C and Dcorrespond to serial dilutions (10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵) of an M.tuberculosis H37Rv settling culture (OD₆₀₀: 0.4).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a pharmaceutical composition comprising acompound preventing EthR from binding to the ethA promoter and athioamide or thiourea.

Preferably, the compound preventing EthR from binding to the ethApromoter is a compound of formula 1

wherein R¹ is optionally substituted phenyl or optionally substitutedpyridyl;

R² is (CH₂), wherein n is 1, 2, 3 or 4;

R³ is CH₃(CH₂)_(m) wherein m is 0, 1, 2 or 3;

X¹ is O, S, NH, N(CH₃) or CH₂; and

X² is O, S or NH.

The pharmaceutical composition of the invention further comprises athioamide or thiourea, which is preferably a compound of formula 2

wherein R⁴ is optionally substituted phenyl, optionally substitutedpyridyl, optionally substituted indolyl, —NR⁷R⁸; or —NH—N═CH—R⁹; R⁵ ishydrogen, C₁-C₆-alkyl, optionally substituted phenyl, optionallysubstituted pyridyl, or a sugar residue;

R⁶ is hydrogen or C₁-C₆-alkyl, or R⁵ and R⁶ together with the N-atom towhich they are bound are pyrrolidine, piperidine or morpholine;

R⁷ is hydrogen, C₁-C₆-alkyl, optionally substituted phenyl, optionallysubstituted pyridyl, or a sugar residue;

R⁸ is hydrogen or C₁-C₆-alkyl, or R⁷ and R⁸ together with the N-atom towhich they are bound are pyrrolidine, piperidine or morpholine; and

R⁹ is optionally substituted phenyl.

Alkyl is in particular C₁-C₆-alkyl, for example C₁-C₄-alkyl, C₁-C₄-Alkylis methyl, ethyl, propyl, e.g. n-propyl or iso-propyl, or butyl, e.g.n-butyl, iso-butyl or tert-butyl, C₁-C₆-Alkyl is methyl, ethyl, propylor butyl as described, or also pentyl, e.g. n-pentyl or iso-pentyl, orhexyl, e.g. n-hexyl or iso-hexyl.

Optionally substituted phenyl is unsubstituted phenyl or phenylsubstituted by one, two or three substituents selected from C₁-C₆-alkyl,e.g. methyl, trifluoromethyl, C₁-C₆-alkoxy, e.g. methoxy, ethoxy oriso-pentoxy, C₁-C₆-alkylcarbonyl, e.g. acetyl, C₁-C₆-alkyl-carbonyloxy,e.g. acetoxy, C₁-C₆-alkylthio, e.g. methylthio, nitro, amino,C₁-C₆-alkylamino, e.g. methylamino or ethylamino, di-C₁-C₆-alkylamino,e.g. dimethylamino or diethylamino, pyrrolidino, piperidino, morpholino,C₁-C₆-alkylcarbonylamino, e.g. acetylamino, and halogen. Halogen isfluoro, chloro, bromo or iodo, particularly fluoro or chloro. Preferablyoptionally substituted phenyl is phenyl or phenyl substituted by one ortwo of the mentioned substituents, in particular one of the mentionedsubstituents in ortho, meta or para position, preferably in meta or paraposition. For example, optionally substituted phenyl is phenyl, methyl-or dimethylphenyl, trifluoromethylphenyl, methoxyphenyl, ethoxyphenyl,acetoxyphenyl, nitrophenyl, dinitrophenyl, aminophenyl,methylamino-phenyl, dimethylaminophenyl, fluorophenyl, chlorophenyl ordichlorophenyl.

Optionally substituted pyridyl is 2-, 3- or 4-pyridyl, unsubstituted orsubstituted by one or two substituents selected from C₁-C₆-alkyl, e.g.methyl or ethyl, trifluoromethyl, C₁-C₆-alkoxy, e.g. methoxy or ethoxy,nitro, amino, C₁-C₆-alkylamino, e.g. methylamino or ethylamino,di-C₁-C₆-alkylamino, e.g. dimethylamino or diethylamino,C₁-C₆-alkyl-carbonylamino, e.g. acetylamino, and halogen. Halogen isfluoro, chloro, bromo or iodo, particularly fluoro or chloro.

Optionally substituted indolyl is 1 H-2-, 3-, 4-, 5-, 6-, or 7-indolyl,unsubstituted or substituted by one or two substituents selected fromC₁-C₆-alkyl, e.g. methyl or ethyl, trifluoromethyl, C₁-C₆-alkoxy, e.g.methoxy or ethoxy, nitro, amino, C₁-C₆-alkylamino, e.g.

methylamino or ethylamino, di-C₁-C₆-alkylamino, e.g. dimethylamino ordiethylamino, C₁-C₆-alkylcarbonylamino, e.g. acetylamino, and halogen.

A sugar residue is

L- or D-furanosyl selected from the aldopentoses arabinose, lyxose,ribose and xylose of formula 3;

L- or D-hexofuranosyl selected from the aldohexoses allose, altrose,glucose, mannose, gulose, idose, galactose and talose of formula 4;

L- or D-hexofuranosyl selected from the ketohexoses fructose, psicose,sorbose and tagatose of formula 5;

L- or D-pyranosyl selected from the aldohexoses allose, altrose,glucose, mannose, gulose, idose, galctose and talose of formula 6; or

L- or D-pyranosyl selected from the ketohexoses fructose, psicose,sorbose and tagatose of formula 7;

in which one, two, three or four hydroxy groups can be methylated,benzylated or acetylated, or one hydroxy group can be replaced byhydrogen, halogen, methylamino, ethylamino, or acetamido.

Particularly preferred in the compound of formula 1 is R¹ with themeaning phenyl. Also particularly preferred in the compound of formula 1is X¹ with the meaning O or CH₂ and X² with the meaning O, i.e. an esteror a ketone.

Most preferred is compound of formula 1 selected from4-phenyl-2-butanone, benzyl acetate, 3-phenylpropyl propionate and2-phenylethyl butyrate, in particular 2-phenylethyl butyrate.

Preferred compounds of formula 2 are those wherein

R⁴ is optionally substituted pyridyl, NR⁷R⁸, or —NH—N═CH—R⁹;

R⁵ is hydrogen, optionally substituted phenyl, or a sugar residue;

R⁶ is hydrogen;

R⁷ is optionally substituted phenyl or a sugar residue;

R⁸ is hydrogen; and

R⁹ is optionally substituted phenyl.

Even more preferred are compounds of formula 2 wherein

R⁴ is substituted pyridyl, NR⁷R⁸, or —NH—N═CH—R⁹;

R⁵ is hydrogen, substituted phenyl, or a sugar residue;

R⁶ is hydrogen;

R⁷ is substituted phenyl or a sugar residue;

R⁸ is hydrogen; and

R⁹ is substituted phenyl.

Most preferred are compounds of formula 2 wherein

R⁴ is pyridyl substituted by C₁-C₆-alkyl, NR⁷R⁸, or —NH—N═CH—R⁹;

R⁵ is hydrogen, phenyl substituted by C₁-C₆-alkoxy, or a sugar residue;

R⁶ is hydrogen;

R⁷ is phenyl substituted by C₁-C₆-alkoxy, or a sugar residue;

R⁸ is hydrogen; and

R⁹ is phenyl substituted by C₁-C₆-alkylcarbonylamino.

Particularly preferred compounds are compounds of formula 2 wherein R⁴is 4-pyridyl substituted by C₁-C₆-alkyl; R⁵ is hydrogen or a sugarresidue; and R⁶ is hydrogen; in particular ethionamide of formula 8:

compounds of formula 2 wherein R⁴ is —NH—N═CH—R⁹; R⁵ is hydrogen or asugar residue;

R⁶ is hydrogen; and R⁹ is phenyl substituted byC₁-C₆-alkylcarbonylamino, in particular thiacetazone of formula 9:

compounds of formula 2 wherein R⁴ is —NR⁷R⁸; R⁵ is phenyl substituted byC₁-C₆-alkoxy;

R⁶ is hydrogen; R⁷ is phenyl substituted by C₁-C₆-alkoxy or a sugarresidue; and R⁸ is hydrogen; in particular isoxyl of formula 10:

or the isoxyl analogN-arabinofuranosyl-N′-[ρ-(isoamyloxy)phenyl]-thiourea of formula 11:

Compounds of formula 1 are known are can be made according to methodswell known in the art.

Compounds of formula 2 are known or can be manufactured as follows:

Thioamides, i.e. compounds of formula 2 wherein R⁴ is optionallysubstituted phenyl, optionally substituted pyridyl or optionallysubstituted indolyl, are obtainable by reacting an amine of formulaHNR⁵R⁶ with a carboxylic acid of formula R⁴—COON to form an amide offormula R⁴—CO—NR⁵R⁶. The amide is reacted with Lawesson's reagent orphosphorus pentasulfide, to obtain the thioamide of formula 2. Anothermethod to synthesize the thioamides is via the Kindler modification ofthe Willgerodt reaction using an aldehyde of formula R⁴—CH═O and anamine of formula HNR⁵R⁶ and react them in the presence of sulphur.

Thioureas, i.e. compounds of formula 2 wherein R⁴ is —NR⁷R⁸, areobtainable by reacting a bromide with potassium thiocyanate to give anisothiocyanate of formula R⁵—N═C═S or R⁷—N═C═S, which is reacted with anamine of formula HNR⁷R⁸ or HNR⁵R⁶, respectively. Correspondinghydrazones, i.e. compounds of formula 2 wherein R⁴ is —NH—N═CH—R⁹, areobtainable by reacting an aldehyde of formula R⁹—CH═O with anhydrazinocarbothioamide of formula R⁵R⁶N—(C═S)—NH—NH₂.

Furthermore the invention relates to a method of screening for compoundspreventing EthR from binding to the ethA promoter. An EthR-basedsynthetic mammalian gene circuit allows to determine, whether a compoundcould be used together with ethionamide in the treatment oftuberculosis.

For the purpose of the invention, EthR is a polypeptide derived from M.tuberculosis EthR [Dover, L. G. et al., J Mol Biol 340, 1095-105(2004)]. By “derived from” is meant, in this context, that thepolypeptide comprises protein domains that contain amino acidsubstitutions, preferably conservative amino acid substitutions, butremain at least 70%, preferably 80%, and more preferably 90% or moreidentical to the naturally occurring M. tuberculosis EthR at the aminoacid level.

“Conservative substitution” is known in the art and is described e.g. byDayhof, M. D., 1978, Nat. Biomed. Res. Found., Washington, D.C., Vol. 5,Sup. 3. Genetically encoded amino acids are generally divided into fourgroups: (1) acidic=aspartate and glutamate; (2) basic=lysine, arginine,and histidine; (3) non-polar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, and tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine, andtyrosine. Phenylalanine, tryptophan and tyrosine are also jointlyclassified as aromatic amino acids. A substitution in a protein of oneamino acid classified in a particular group by another amino acid in thesame group is generally regarded as a conservative substitution.

For the purpose of the invention, O_(ethR) is a polynucleotide relatedto the M. tuberculosis ethA promoter, which is defined inEngohang-Ndong, J. et al., Mol Microbiol 51, 175-88 (2004). By anO_(ethR) sequence “related to” the M. tuberculosis ethA promoter ismeant, in this context, that the polynucleotide sequence of O_(ethR)contains base changes or modified nucleotides compared to the naturallyoccurring polynucleotide, but still can bind the EthR eitherconstitutively or in a small molecule-dependent way.

Structural analysis classifying EthR as a TetR/CamR family repressorsuggests the existence of compounds that could modulate the affinity ofEthR for its O_(ethR) operator [Dover, L. G. et al., J Mol Biol 340,1095-105 (2004)]. Following a synthetic biology approach a gene networkwhose topology enables detection of EthR-binding molecules inside humancells is designed, thereby scoring for non-cytotoxic and bioavailablecompounds accessing the pathogenic habitat of M. tuberculosis (FIG. 1A).The gene circuit consists of a synthetic transactivator, EthR fused tothe VP16 transactivation domain of Herpes simplex (pWW489,P_(sv40)-EthR-VP16-pA), which induces SEAP (human placental secretedalkaline phosphatase) expression in human embryonic kidney cells(HEK-293) after binding to a chimeric promoter containing O_(ethR) 5′ ofa minimal Drosophila heat shock protein 70 promoter (P_(hsp90min);pWW491, O_(ethR)-P_(hsp70min)-SEAP-pA, 8.2±0.8 U/L; background level ofpWW491 0.4±0.1 U/L, FIG. 1 a). Cell-permeable EthR-interacting compoundsare expected to release EthR-VP16 from O_(ethR)-P_(hsp70min), therebyrepressing SEAP production to basal levels (FIG. 1A). Interestingly,hexadecyl octanoate (10 mM; ClogP=11.29), identified in cristallographystudies to compromise EthR's DNA-binding capacity [Frenois, F. et al.,Mol Cell 16, 301-7, 2004], fails to decrease SEAP expression in HEK-293containing pWW489 and pWW491, suggesting that it remains a non-trivialchallenge to develop bioavailable EthR-binding compounds.

In more general terms, the invention relates to a method of screeningfor compounds preventing EthR from binding to the O_(ethR) operator. Inparticular, the O_(ethR) operator or a derivative thereof isfunctionally linked to a mammalian cell-compatible promoter in a way,that binding of EthR to O_(ethR) modulates the transcriptional activityof said mammalian cell-compatible promoter. The transcriptional activityof said mammalian cell-compatible promoter can be analyzed by placing asuitable reporter gene (e.g. human placental secreted alkalinephosphatase (SEAP), a fluorescent protein or a luciferase) under thecontrol of said promoter. In order to enhance the transcriptionalactivity-modulating effect of EthR, it can optionally be functionallylinked to a transactivator or transrepressor domain (a non-limitingselection of which are cited in U.S. Pat. No. 6,287,813). The screeningis performed by contacting a test compound with a mammalian cellharbouring said mammalian cell-compatible promoter functionally linkedto O_(ethR) and a suitable reporter gene and further harbouring EthRoptionally fused to a transactivator or transrepressor domain. A changein reporter gene expression indicates that the test compound mightinterfere with binding of EthR to its operator O_(ethR).

In the method of screening for compounds preventing EthR from binding tothe ethA promoter of the invention, compounds are tested in mammaliancells comprising the O_(ethR) operator or a derivative thereoffunctionally linked to a mammalian cell-compatible promoter in a way,that binding of EthR to O_(ethR) modulates the transcriptional activityof said mammalian cell-compatible promoter, and the test compound isdetermined to be a compound preventing EthR from binding to the ethApromoter if it represses expression of a reporter gene under control ofsaid mammalian cell-compatible promoter.

Capitalizing on cristallography data [Dover, L. G. et al., J Mol Biol340, 1095-105 (2004)] describing EthR's small-molecule binding site as“hydrophobic tunnel-like cavity fitting a lipophilic ligand” and on theobservation that repressors are often feed-back controlled by thesubstrates or products of their target gene, a library of hydrophilicketones and esters with ClogP values below 4 to enable screening inaqueous solutions (Table 1) is analyzed.

TABLE 1 Compound Structure Formula M_(w) C_(logP) Methyl hexanoate

C₇H₁₄O₂ 130.18 2.298 Butyl propionate

C₇H₁₄O₂ 130.18 2.298 Pentyl acetate

C₇H₁₄O₂ 130.18 2.298 4-Phenyl-2-butanone

C₁₀H₁₂O 148.20 1.889 Methyl phenylacetate

C₉H₁₀O₂ 150.17 1.820 Benzyl acetate

C₉H₁₀O₂ 150.17 1.960 2-Phenylethyl acetate

C₁₀H₁₂O₂ 164.20 2.279 2-Phenylethyl propionate

C₁₁H₁₄O₂ 178.23 2.808 2-Phenylethyl butyrate

C₁₂H₁₆O₂ 192.25 3.337 3-Phenylpropyl propionate

C₁₂H₁₆O₂ 192.25 3.187 2-Phenylethyl isopentanoate

C₁₃H₁₈O₂ 206.28 3.736 Hexadecyl octanoate

C₂₄H₄₈O₂ 368.64 11.29

When HEK-293 populations containing the EthR-based gene circuit areexposed to 0-3.2 mM of individual library components, only4-phenyl-2-butanone, benzyl acetate, 3-phenylpropyl propionate and2-phenylethyl butyrate induce a significant decrease in SEAP expression.As the SEAP production decline does not correlate with cytotoxicity(assessed by using HEK-293 engineered for isogenic constitutive SEAPexpression) these compounds may release EthR-VP16 fromO_(etR)-P_(hsp90min) (FIG. 1B).

2-Phenylethyl butyrate (IC₅₀=0.5 mM) is preferred, since it does notshow cytotoxity and demonstrates reasonable activity (FIG. 1B).Furthermore, 2-phenylethyl butyrate is a licensed food additive, seeJoint FAO/WHO Expert Committee on Food Additives, JECFA No. 991.

2-Phenylethyl butyrate retains its regulating activity in vivo.Microencapsulated _(EthR)HEK-SEAP (transgenic for pWW489 and pWW491; seeFIGS. 1C and 1D for clonal variation, FIG. 1E for adjustability and FIG.1F for reversibility of the gene circuit) is implanted intraperitoneallyinto mice which are subsequently injected with 2-phenylethyl butyrate(625 μl/kg). 2-Phenylethyl butyrate significantly reduces SEAP serumlevels of treated mice (with 2-phenylethyl butyrate 0.21±0.03 U/L;without 2-phenylethyl butyrate: 0.42±0.04 U/L)) suggesting that thiscompound is bioavailable and reaching EthR-inactivating concentrationsinside target cells.

Escherichia coli, engineered for EthR-controlled GFP expression showsadjustable green fluorescence when exposed to 2-phenylethyl butyratelevels previously used in vivo, indicating that this compound alsotriggers release of EthR from O_(ethR) in prokaryotes (FIGS. 2A, 2C and2D showing FACS parameters). Indeed, when using an ELISA combiningimmobilized O_(ethR) with His₆-tagged EthR (FIG. 2B) (or E¹⁷ and ETR asspecificity control, FIG. 3) produced in E. coli it is confirmed that2-phenylethyl butyrate exclusively and specifically modulates theEthR-O_(ethR) interaction.

The repressor protein E binds to its cognate operator sequence ETR andis released thereof in the presence of macrolide antibiotics likeerythromycin as described in U.S. Pat. No. 7,273,723.

Growth of M. tuberculosis is significantly impaired in the presence ofethionamide due to EthA-mediated conversion of this prodrug into anantimycobacterial nicotinamide adenine dinucleotide derivative.EthR-mediated repression of ethA transcription requires rather highclinical doses of ethionamide (up to 1 g/day, Holdiness, M. R., ClinPharmacokinet 9, 511-44 (1984)) which is associated with severeside-effects including neurotoxicity and fatal hepatotoxicity, yet isoften still insufficient to reach minimum inhibitory levels in the bloodstream. Therefore, 2-phenylethyl butyrate-triggered dissociation of EthRfrom the ethA promoter resulting in derepression of ethA increases thesensitivity of Mycobacterium to ethionamide-based therapy. Growth of M.bovis BCG and M. tuberculosis H37Rv in the presence of sub-inhibitoryethionamide concentrations (0.25 and 0.5 μg/ml), which are easilyreached by therapeutic doses, (c_(max) (250 mg oral)=2 μg/ml, t_(1/2)=2h) is dose-dependently inhibited by 0.5 and 2 mM 2-phenylethyl butyrate.Since 2-phenylethyl butyrate alone does not show any growth inhibitoryeffect it must act synergistically with ethionamide to kill the pathogen(FIG. 4).

Pharmaceutical Compositions

The invention relates to a pharmaceutical composition comprising acompound preventing EthR from binding to the ethA promoter and athioamide or thiourea. In particular the invention relates to such acomposition comprising a compound of formula 1 as defined hereinbefore,preferably selected from 4-phenyl-2-butanone, benzyl acetate,3-phenylpropyl propionate and 2-phenylethyl butyrate, in particular2-phenylethyl butyrate, and a compound of formula 2 as definedhereinbefore, preferably ethionamide.

As a consequence of adding a compound preventing EthR from binding tothe ethA promoter to a pharmaceutical composition useful in thetreatment of tuberculosis, the dose of the thioamide or thiourea, e.g.of ethionamide, may be substantially reduced, reducing thereby the knownside effects without reducing its efficacy.

The pharmaceutical compositions of the invention are not only useful forthe treatment of tuberculosis, i.e. a disease caused by Mycobacteriumtuberculosis, but also for the treatment of diseases caused by relatedbacteria with EthR related proteins binding to the corresponding ethArelated promoter, in particular Mycobacterium leprae, Mycobacteriumulcerans, Mycobacterium marinum, Mycobacterium sp. MCS, Mycobacteriumsp. KMS, Mycobacterium sp. JLS, Mycobacterium vanbaalenii, Mycobacteriumavium subsp. paratuberculosis, Mycobacterium avium, Mycobacteriumsmegmatis, Mycobacterium gilvum, Mycobacterium abscessus, Acinetobacterbaumannii, Renibacterium salmoninarum, Mycobacterium gilvum,Streptococcus pyogenes, Bacillus licheniformis, Clostridium spiroforme,and Bacillus anthracis. Such diseases are leprosy, buruli-ulcer disease,atypical mycobacteriosis, Johne's and Crohn's disease, hot tub lung,lady

Windermere syndrome, chronic lung disease, post-traumatic woundinfections, post-tympanostomy tube otorrhea, disseminated cutaneousdiseases, Acinetobacter baumanii caused infections, pharyngitis,impetigo, erysipelas, cellulitis, necrotizing fasciitis, scarlet fever,toxic shock septicaemia, peritonitis, ophthalmitis, diarrhoea andsplenic fever. Pharmaceutical compositions according to the inventionare compositions for enteral administration, such as nasal, buccal,rectal or, especially, oral administration, and for parenteraladministration, such as intravenous, intramuscular or subcutaneousadministration. The compositions comprise the thioamide or thiourea,e.g. ethionamide, and the compound preventing EthR from binding to theethA promoter alone or, preferably, together with a pharmaceuticallyacceptable carrier. The dosage of the active ingredients depends uponthe patient, its age, weight, and individual condition, the individualpharmacokinetic data, and the mode of administration.

The invention relates also to pharmaceutical compositions for use in amethod for the prophylactic or especially therapeutic management of thehuman or animal body, in particular in a method of treatingtuberculosis, leprosy, buruli-ulcer disease, atypical mycobacteriosis,Johne's and Crohn's disease, hot tub lung, lady Windermere syndrome,chronic lung disease, post-traumatic wound infections, post-tympanostomytube otorrhea, disseminated cutaneous diseases, Acinetobacter baumaniicaused infections, pharyngitis, impetigo, erysipelas, cellulitis,necrotizing fasciitis, scarlet fever, toxic shock septicaemia,peritonitis, ophthalmitis, diarrhoea and splenic fever.

The invention relates also to processes and to the use of compoundspreventing EthR from binding to the ethA promoter for the manufacture ofpharmaceutical preparations which further comprise a thioamide orthiourea, e.g. ethionamide, as active component.

The pharmaceutical compositions comprise from approximately 5% toapproximately 95% of a mixture of a thioamide or thiourea, e.g. ofethionamide, and of a compound preventing EthR from binding to the ethApromoter in relative molar amounts of between 1:1 up to 1:10,000,preferably 1:10 up to 1:5000. Single-dose administration forms comprisefrom approximately 20% to approximately 90% of the mentioned mixture,and forms that are not of single-dose type from approximately 5% toapproximately 20% of the mentioned mixture. Unit dose forms are, forexample, coated and uncoated tablets, ampoules, vials, suppositories, orcapsules. Further dosage forms are, for example, ointments, creams,pastes, foams, tinctures, lip-sticks, drops, syrups, sprays, and thelike. Examples are capsules containing from about 0.05 g to about 1.0 gof a mixture of the active ingredients.

It is also possible to use the mixture of a thioamide or thiourea, e.g.of ethionamide, and of a compound preventing EthR from binding to theethA promoter in two separate pharmaceutical unit dose forms, and such acombination is also part of the present invention. For example, athioamide or thiourea, e.g. ethionamide, may be used in amounts of 0.01g to about 0.5 g, e.g. in commercially available dose forms comprisingfrom 0.05 g to about 0.5 g ethionamide, in combination with a differentor same dose form comprising the compound preventing EthR from bindingto the ethA promoter, for example 2-phenylethyl butyrate, in amounts of0.5 g to about 5.0 g, as a kit of parts.

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example by means of conventional mixing,granulating, coating, dissolving, emulsifying or lyophilizing processes.Optionally, the compound preventing

EthR from binding to the ethA promoter, e.g. 2-phenylethyl butyrate, canbe formulated in liposomes.

For parenteral administration solutions of the active ingredients arepreferred, and also suspensions, emulsions or dispersions, especiallyisotonic aqueous solutions, dispersions, emulsions or suspensions which,for example in the case of lyophilized compositions comprising theactive ingredients alone or together with a carrier, for examplemannitol, can be made up before use. The pharmaceutical compositions maybe sterilized and/or may comprise excipients, for example preservatives,stabilizers, wetting agents and/or emulsifiers, solubilizers, salts forregulating osmotic pressure and/or buffers and are prepared in a mannerknown per se, for example by means of conventional dissolving andlyophilizing processes. The said solutions or suspensions may compriseviscosity-increasing agents, typically sodium carboxymethylcellulose,carboxymethyl-cellulose, dextran, polyvinylpyrrolidone, or gelatins, oralso solubilizers, e.g. Tween 80® (polyoxyethylene(20)sorbitanmono-oleate).

Suspensions in oil comprise as the oil component the vegetable,synthetic, or semi-synthetic oils customary for injection purposes. Inrespect of such, special mention may be made of liquid fatty acid estersthat contain as the acid component a long-chained fatty acid having from8 to 22, especially from 12 to 22, carbon atoms. The alcohol componentof these fatty acid esters has a maximum of 6 carbon atoms and is amonovalent or polyvalent, for example a mono-, di- or trivalent,alcohol, especially glycol and glycerol. As mixtures of fatty acidesters, vegetable oils such as cottonseed oil, almond oil, olive oil,castor oil, sesame oil, soybean oil and groundnut oil are especiallyuseful.

The manufacture of injectable preparations is usually carried out understerile conditions, as is the filling, for example, into ampoules orvials, and the sealing of the containers.

Suitable carriers for oral compositions are especially fillers, such assugars, for example lactose, saccharose, mannitol or sorbitol, cellulosepreparations, and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, and also binders, such asstarches, for example corn, wheat, rice or potato starch,methylcellulose, hydroxypropyl methylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl-pyrrolidone, and/or, ifdesired, disintegrators, such as the above-mentioned starches, alsocarboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid ora salt thereof, such as sodium alginate. Additional excipients areespecially flow conditioners and lubricants, for example silicic acid,talc, stearic acid or salts thereof, such as magnesium or calciumstearate, and/or polyethylene glycol, or derivatives thereof.

Tablet cores can be provided with suitable, optionally enteric, coatingsthrough the use of, inter alia, concentrated sugar solutions which maycomprise gum arabic, talc, polyvinyl-pyrrolidone, polyethylene glycoland/or titanium dioxide, or coating solutions in suitable organicsolvents or solvent mixtures, or, for the preparation of entericcoatings, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropyl-methylcellulose phthalate.Dyes or pigments may be added to the tablets or tablet coatings, forexample for identification purposes or to indicate different doses ofactive ingredient.

Pharmaceutical compositions for oral administration also include hardcapsules consisting of gelatin, and also soft, sealed capsulesconsisting of gelatin and a plasticizer, such as glycerol or sorbitol.The hard capsules may contain the active ingredient in the form ofgranules, for example in admixture with fillers, such as corn starch,binders, and/or glidants, such as talc or magnesium stearate, andoptionally stabilizers. In soft capsules, the active ingredient ispreferably dissolved, emulsified or suspended in suitable liquidexcipients, such as fatty oils, paraffin oil or liquid polyethyleneglycols or fatty acid esters of ethylene or propylene glycol, to whichstabilizers and detergents, for example of the polyoxyethylene sorbitanfatty acid ester type, may also be added.

Pharmaceutical compositions suitable for rectal administration are, forexample, suppositories that consist of a combination of the activeingredient and a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols.

The present invention relates furthermore to a method for the treatmentof tuberculosis and related diseases, which comprises administering amixture of a thioamide or thiourea, e.g. ethionamide, and of a compoundpreventing EthR from binding to the ethA promoter, for example2-phenylethyl butyrate, in a quantity effective against said disease, toa warm-blooded animal requiring such treatment. The mixture can beadministered in the form of pharmaceutical compositions comprising themixture, or also the components separately at the same time or atdifferent times within the day, prophylactically or therapeutically,preferably in an amount effective against the tuberculosis or relateddisease, to a warm-blooded animal, for example a human, requiring suchtreatment. In the case of an individual having a bodyweight of about 70kg the daily dose of the mixture administered is from approximately 0.01g to approximately 50 g, preferably from approximately 0.05 g toapproximately 10 g, of a mixture containing the components in relativeamounts of between 1:1 and 1:10,000.

The present invention relates especially also to the use of a compoundpreventing EthR from binding to the ethA promoter, for example acompound of formula 1 as defined hereinbefore, such as4-phenyl-2-butanone, benzyl acetate, 3-phenylpropyl propionate and2-phenylethyl butyrate, in particular of 2-phenylethyl butyrate, as suchor in the form of a pharmaceutical formulation with at least onepharmaceutically acceptable carrier for the therapeutic and alsoprophylactic management of tuberculosis in combination with a thioamideor thiourea, for example a compound of formula 2, such as ethionamide,administered either separately or in a fixed combination. The preferreddose quantity, composition, and preparation of pharmaceuticalformulations which are to be used in each case are described above.

The following Examples serve to illustrate the invention withoutlimiting the invention in its scope.

Examples

Vector design.

pWW489 (P_(sv40)-ethR-vp16-pA) is constructed by PCR-mediatedamplification of ethR from genomic M. bovis DNA using oligonucleotidesOWW400 (5′-gcatccatatgaattccaccatg accacctccgcggcca-3′) and OWW401(5′-cgatcgcgcgcggctgtacgcggagcggttctcgccgtaaatgc-3′) followed byrestriction and ligation (EcoRI/BssHII) into pWW35 [Weber, W. et al.,Nat Biotechnol 20, 901-7 (2002)]. pWW491 (O_(ethR)-P_(hsp70min)-SEAP-pA)is obtained by direct cloning of a synthetic O_(ethR) sequence(5′-gacgtcgatccacgctatcaacgtaatgtcgaggccgtcaacgagatgtcgacactatcgacacgtagcctgcagg-3′) (AatII/SbfI) into pMF172 [Weber, W.et al., supra]. pWW488 (P_(T7)-ethR-vp16-his₆) is constructed byPCR-mediated amplification of ethR-vp16 from pWW489 usingoligonucleotides OWW400 and OWW60 (5′-gctctagagcaagcttttaatggtgatggtgatgatgcccaccgtactgtcaattccaag-3′) followed by cloning(NdeI/HindIII) into pRSETmod [Weber, C. C. et al., Biotechnol Bioeng 89,9-17 (2005)]. pWW856 (P_(ethR)-gfp-pA) is constructed in three steps:(i) gfp is PCR-amplified from pLEGFP-N1 (Clontech, Palo Alto, Calif.,USA) using oligonucleotides OWW848 (5′-ggcttgaattcaaaggagatataccatggtgagcaagggcgag-3′) and OWW849(5′-ggctttctagacaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttacttgtacagctcgtccatgccg-3′) and cloned (EcoRI/XbaI) intopWW56 [Weber W. et al., supra] (pWW854). (ii) A synthetic O_(ethR)sequence is directly cloned (HindIII/l EcoRI) into pWW854 (pWW855).(iii) P_(ethR)-gfp is excised (BamHI/StuI) from pWW855 and ligated(BamHI/ScaI) into pACYC177 (NEB, Ipswich, Mass., USA) (pWW856). pWW862(P-_(T7)-ethR-his₆) is assembled by annealing oligonucleotides OWW479(5′-cgcgcatcatcatcat catcattaagcggccgca-3′) and OWW480(5′-agcttgcggccgcttaatgatgatgatgatgatg-3′) and cloning thedouble-stranded DNA BssHII/HindIII into pWW488. pWW871(5′LTR-Ψ⁺-ethR-vp16-P_(pGK)-neo^(R)-3′LTR) was designed by cloningethR-vp16 of pWW489 (EcoRI/BamHI) into pMSCVneo (Clontech). pWW35(P_(sv40)-E-vp16-pA), pWW37 (ETR-P_(hcMVmin)-seap-pA) and pWW313(P_(T7)-E-his₆-pA) are described in Weber, C. C. et al., supra.

Cell culture.

Human embryonic kidney cells (HEK-293, ATCC CRL-1573) are cultivated inDulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, Calif.,USA) supplemented with 10% fetal calf serum (Pan Biotech GmbH,Aidenbach, Germany, cat. no. 3302, lot P231902) and 1% of apenicillin/streptomycin solution (Sigma, St. Louis, Mo., USA, cat. no.4458). Cells are transfected using standard calcium phosphate procedures[Weber W. et al., supra] and retroviral particles are produced accordingto the manufacturer's protocol (Clontech). _(EthR)HEK, transgenic forconstitutive EthR-VP16 expression, is constructed by transducing HEK-293with pWW871-derived retroviral particles followed by selection in DMEMcontaining 200 μg/ml neomycin and single-cell cloning. Cotransfection of_(EthR)HEK with pWW491 and pPUR (Clontech), subsequent selection in 200μg/ml neomycin, 1 μg/ml puromycin followed by single-cell cloningresulted in _(EthR)HEK⁻SEAP. SEAP production is quantified as describedin Schlatter, S. et al., Gene 282, 19-31 (2002).

Chemicals.

Pentyl acetate, methylphenyl acetate, 2-phenylethyl acetate,4-phenyl-2-butanone (all Fluka) and 2-phenylethyl butyrate (Sigma) arecommercially obtained. Methyl hexanoate is obtained by reacting hexanoicacid with thionylchloride in methanol. Butyl propionate, 2-phenylethylpropionate, 2-phenylethyl isopentanoate and 3-phenylpropyl propionateare prepared by reacting the corresponding alcohol with the acidchloride in dichloromethane using triethylamine as a base. Hexadecyloctanoate and benzyl-acetate are synthesized from the bromide and theacid with K₂CO₃ as the base in dimethylformamide. All esters arepurified either by column chromatography (silica, ethylacetate/hexane)or distillation. ClogP is determined using Chemdraw Ultra 10.0(CambridgeSoft, Cambridge, Mass., USA). Erythromycin (Sigma, St. Louis,Mo., USA, cat. no. E5389) is used as a 1,000× stock solution of 5 mg/mlin ethanol. Ethionamide is purchased from Sigma (cat. no. E6005) andprepared as 200× stock solution in DMSO.

FACS analysis.

E. coli BL21(DE3) (Invitrogen), transformed with pWW488 and pWW856 orpWW856 alone, are grown overnight in Luria Bertani (LB) media containing100 μg/ml ampicillin and 30 μg/ml kanamycin (for pWW856-transformedcells only). Then, 150 of E. coli suspension (OD₆₀₀ 1.3) is added to 2ml fresh LB media containing antibiotics and 1 mM IPTG where indicated.After growth for 5.5 h at 37° C., 500 μl of the suspension istransferred to a new tube and centrifuged for 3 min at 800× g. Thepellet is washed twice with 1 ml PBS and resuspended in 2 ml PBS forFACS analysis (>10,000 cells/sample), which is performed on a CytomicsFC500 (Beckman Coulter, Fullerton, Calif.) with 405 nm used forexcitation and 510 nm for emission. FACS gates are shown inSupplementary FIG. 2. ELISA.

EthR-his₆ is produced as previously described for E-his₆ [Weber, C. C.et al., supra] with the exception that the biotinylated operatorsequence (O_(ethR)) is incubated with a 100 μl streptavidin agarose beadsuspension (Novagen, Madison, Wis., USA, cat. no. 69203) for 1 h at 4°C. while rotating.

In vivo methods.

_(EthR)HEK-SEAP is encapsulated in alginate-poly-L-lysine-alginatecapsules (200 cells/capsule) as described previously [Weber W. et al.,supra]. Mice are injected intraperitoneally with 700 μl capsulesuspension containing 2×10⁶ cells. One and 25 hours post capsuleimplantation, the mice are injected with 2-phenylethyl butyrate at theindicated concentration (the injection volume is adjusted to 100 μl byadding canola oil (Migros, Zurich, Switzerland)). 48 hours post capsuleimplantation, serum samples are analyzed for SEAP expression. Dissectionof the animals reveals no inflammation at the injection site.

Mycobacteria cultivation and susceptibility testing.

M. tuberculosis H37Rv (ATCC27294) and M. bovis BCG #1721, astreptomycin-resistant derivative of BCG Pasteur, carrying anon-restrictive rpsL mutation (K42R) [Sander, P. et al., Mol Microbiol52, 1543-52 (2004)] are grown in Middlebrook 7H9 supplemented with oleicacid, albumin, dextrose, catalase (Difco) and Tween 80 (0.05%) untilmid-log phase. Ten-fold serial dilutions (20 μl) are streaked onMiddlebrook 7H10-OADC agar plates containing solvent (DMSO, 200-folddilution), ethionamide (0.25-0.5 μg/ml) and 2-phenylethyl butyrate (0.5or 2 mM) where indicated. Plates are incubated at 37° C. and growth isdocumented after 2 and 3 weeks.

1. A pharmaceutical composition comprising a compound preventing EthRfrom binding to the ethA promoter and a thioamide or thiourea.
 2. Thepharmaceutical composition according to claim 1, wherein the thioamideor thiourea is ethionamide.
 3. The pharmaceutical composition accordingto claim 1, wherein the compound preventing EthR from binding to theethA promoter is a compound of formula 1

wherein R¹ is optionally substituted phenyl or optionally substitutedpyridyl; R² is (CH₂)_(n) wherein n is 1, 2, 3 or 4; R³ is CH₃(CH₂)_(m)wherein m is 0, 1, 2 or 3; X¹ is O, S, NH, N(CH₃) or CH₂; and X² is O, Sor NH.
 4. The pharmaceutical composition according to claim 3, whereinin the compound of formula 1 R¹ is unsubstituted phenyl, phenylsubstituted by one, two or three substituents selected from C₁-C₆-alkyl,trifluoromethyl, C₁-C₆-alkoxy, C₁-C₆-alkylcarbonyl,C₁-C₆-alkylcarbonyloxy, C₁-C₆-alkylthio, nitro, amino, C₁-C₆-alkylamino,di-C₁-C₆-alkylamino, pyrrolidino, piperidino, morpholino,C₁-C₆-alkylcarbonylamino, and halogen, unsubstituted 2-, 3- or4-pyridyl, or 2-, 3- or 4-pyridyl substituted by one or two substituentsselected from C_(i)-C₆-alkyl, trifluoromethyl, C_(i)-C₆-alkoxy, nitro,amino, C_(i)-C₆-alkylamino, di-C₁-C₆-alkylamino,C₁-C₆-alkylcarbonylamino, and halogen.
 5. The pharmaceutical compositionaccording to claim 4, wherein, in the compound of formula 1, R₁ isphenyl optionally substituted by one, two or three substituents selectedfrom methyl, trifluoromethyl, methoxy, ethoxy, nitro, amino,methylamino, dimethylamino, ethylamino, diethylamino and halogen, or 2-,3- or 4-pyridyl optionally substituted by one or two substituentsselected from methyl, trifluoromethyl, methoxy, ethoxy, nitro, amino,methylamino, dimethylamino, ethylamino, diethylamino and halogen.
 6. Thepharmaceutical composition according to claim 1, wherein the compoundpreventing EthR from binding to the ethA promoter is selected from4-phenyl-2-butanone, benzyl acetate, 3-phenylpropyl propionate and2-phenylethyl butyrate.
 7. The pharmaceutical composition according toclaim 1, wherein the compound preventing EthR from binding to the ethApromoter is 2-phenylethyl butyrate.
 8. The pharmaceutical compositionaccording to claim 1, wherein the thioamide or thiourea is a compound offormula 2

wherein R⁴ is optionally substituted phenyl, optionally substitutedpyridyl, optionally substituted indolyl, —NR⁷R⁸; or —NH—N═CH—R⁹; R⁵ ishydrogen, C_(i)-C₆-alkyl, optionally substituted phenyl, optionallysubstituted pyridyl, or a sugar residue; R⁶ is hydrogen or C₁-C₆-alkyl,or R⁵ and R⁶ together with the N-atom to which they are bound arepyrrolidine, piperidine or morpholine; R⁷ is hydrogen, C₁-C₆-alkyl,optionally substituted phenyl, optionally substituted pyridyl, or asugar residue; R⁸ is hydrogen or C₁-C₆-alkyl, or R⁷ and R⁸ together withthe N-atom to which they are bound are pyrrolidine, piperidine ormorpholine; and R⁹ is optionally substituted phenyl.
 9. Thepharmaceutical composition according to claim 8, wherein, in thecompound of formula 2, R⁴ is optionally substituted pyridyl, NR⁷R⁸, or—NH—N═CH—R⁹; R⁵ is hydrogen, optionally substituted phenyl, or a sugarresidue; R⁶ is hydrogen; R⁷ is optionally substituted phenyl or a sugarresidue; R⁸ is hydrogen; and R⁹ is optionally substituted phenyl. 10.The pharmaceutical composition according to claim 8, wherein, in thecompound of formula 2, R⁴ is pyridyl substituted by C₁-C₆-alkyl, NR⁷R⁸,or —NH—N═CH—R⁹; R⁵ is hydrogen, phenyl substituted by C₁-C₆-alkoxy, or asugar residue; R⁶ is hydrogen; R⁷ is phenyl substituted by C₁-C₆-alkoxy,or a sugar residue; R⁸ is hydrogen; and R⁹ is phenyl substituted byC₁-C₆-alkylcarbonylamino.
 11. The pharmaceutical composition accordingto claim 8, wherein, in the compound of formula 2, R⁴ is 4-pyridylsubstituted by C₁-C₆-alkyl; R⁵ is hydrogen or a sugar residue; and R⁶ ishydrogen; or R⁴ is —NH—N═CH—R⁹; R⁵ is hydrogen or a sugar residue; R⁶ ishydrogen; and R⁹ is phenyl substituted by C₁-C₆-alkylcarbonylamino; orR⁴ is —NR⁷R⁸; R⁵ is phenyl substituted by C₁-C₆-alkoxy; R⁶ is hydrogen;R⁷ is phenyl substituted by C₁-C₆-alkoxy or a sugar residue; and R⁸ ishydrogen.
 12. The pharmaceutical composition according to claim 8,wherein the compound of formula 2 is thiacetazone, isoxyl orN-arabinofuranosyl-N′-[p-(isoamyloxy)phenyl]-thiourea.
 13. Thepharmaceutical composition according to claim 1, wherein the relativemolar amounts of the thioamide or thiourea and the compound preventingEthR from binding to the ethA promoter is between 1:1 and 1:10,000. 14.The pharmaceutical composition according to claim 1, wherein thecomposition consists of two separate dose forms containing the thioamideor thiourea and the compound preventing EthR from binding to the ethApromoter, respectively.
 15. A method for the treatment of tuberculosisand related diseases selected from leprosy, buruli-ulcer disease,atypical mycobacteriosis, Johne's and Crohn's disease, hot tub lung,lady Windermere syndrome, chronic lung disease, post-traumatic woundinfections, post-tympanostomy tube otorrhea, disseminated cutaneousdiseases, Acinetobacter baumanii caused infections, pharyngitis,impetigo, erysipelas, cellulitis, necrotizing fasciitis, scarlet fever,toxic shock septicaemia, peritonitis, ophthalmitis, diarrhoea andsplenic fever, which comprises administering a mixture of a thioamide orthiourea and of a compound preventing EthR from binding to the ethApromoter in a quantity effective against said disease, to a warm-bloodedanimal requiring such treatment.
 16. A mixture of a thioamide orthiourea and of a compound preventing EthR from binding to the ethApromoter for the treatment of tuberculosis and related diseases selectedfrom leprosy, buruli-ulcer disease, atypical mycobacteriosis, Johne'sand Crohn's disease, hot tub lung, lady Windermere syndrome, chroniclung disease, post-traumatic wound infections, post-tympanostomy tubeotorrhea, disseminated cutaneous diseases, Acinetobacter baumanii causedinfections, pharyngitis, impetigo, erysipelas, cellulitis, necrotizingfasciitis, scarlet fever, toxic shock septicaemia, peritonitis,ophthalmitis, diarrhoea and splenic fever.
 17. A mixture according toclaim 16 of ethionamide and of a compound preventing EthR from bindingto the ethA promoter for the treatment of tuberculosis.
 18. A method ofscreening for compounds preventing EthR from binding to the ethApromoter, wherein compounds are tested in mammalian cells comprising theO_(ethR) operator or a derivative thereof functionally linked to amammalian cell-compatible promoter in a way, that binding of EthR toO_(ethR) modulates the transcriptional activity of said mammaliancell-compatible promoter, and the test compound is determined to be acompound preventing EthR from binding to the ethA promoter if itrepresses expression of a reporter gene under control of said mammaliancell-compatible promoter.